Comparison of uridine and N1-methylpseudouridine mRNA platforms in development of an Andes virus vaccine

The rodent-borne Andes virus (ANDV) causes a severe disease in humans. We developed an ANDV mRNA vaccine based on the M segment of the viral genome, either with regular uridine (U-mRNA) or N1-methylpseudouridine (m1Ψ-mRNA). Female mice immunized by m1Ψ-mRNA developed slightly greater germinal center (GC) responses than U-mRNA-immunized mice. Single cell RNA and BCR sequencing of the GC B cells revealed similar levels of activation, except an additional cluster of cells exhibiting interferon response in animals vaccinated with U-mRNA but not m1Ψ-mRNA. Similar immunoglobulin class-switching and somatic hypermutations were observed in response to the vaccines. Female Syrian hamsters were immunized via a prime–boost regimen with two doses of each vaccine. The titers of glycoprotein-binding antibodies were greater for U-mRNA construct than for m1Ψ-mRNA construct; however, the titers of ANDV-neutralizing antibodies were similar. Vaccinated animals were challenged with a lethal dose of ANDV, along with a naïve control group. All control animals and two animals vaccinated with a lower dose of m1Ψ-mRNA succumbed to infection whereas other vaccinated animals survived without evidence of virus replication. The data demonstrate the development of a protective vaccine against ANDV and the lack of a substantial effect of m1Ψ modification on immunogenicity and protection in rodents.


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For in-vitro studies, biological replicates were used as the minimum suitable for statistical comparisons.Deviations in these triplicates were limited and statistical support significant.Syrian hamsters demonstrate 100% mortality when infected with high doses of ANDV.This is what we expect in our control group.However, vaccine efficiency may vary, particularly depending on dose and regimen of administration.Group sample sizes of 5 in experimental and control groups achieve 83% power to detect a difference between the group proportions of 0.849.The proportion in the treatment group is assumed to be 0.001 under the null hypothesis and 0.85 under the alternative hypothesis.The proportion in the control group is 0.001.The test statistic used is the two-sided Fisher's Exact Test.The significance level of the test is targeted at 0.05.
For the mouse study of GC development, group sample sizes of 9 achieve 89.29% power to reject the null hypothesis of equal means when the population mean difference is "1 -"2 = 1.6 -0 = 1.6 with a standard deviation for both groups of 1 and with a significance level (alpha) of 0.05 using a two-sided equal-variance t-test.For B-cell repertoire sequencing we used mice with group size of 4, mainly because of very high costs of this study.At 95% confidence level (2tailed unpaired T-test) a sample size of 4 animals per group at each time point will provide greater than 80% power to detect an increase in mean of the log transformed measure of 2 assuming a standard deviation of 0.8.
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All experiments were done in biological triplicates as the minimum, or on groups of 4 to 18 animals.Some of the animal studies were done as 2-3 independent identical experiments (to limit animal number per each experiment to make it doable).Electron microscopy experiment was performed twice.The details are provided in figure captions.All results were reproducible, as also demonstrated by statistical analysis.
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Reference sequence of ANDV segment M used for the development of mRNA is available in GenBank under accession code NC_003467 [https:// www.ncbi.nlm.nih.gov/nuccore/NC_003467]; the same virus was used in our in-vitro and in-vivo studies.Sequence of SNV used in the in-vitro studies is available under accession number AF281850 [https://www.ncbi.nlm.nih.gov/nuccore/AF281850].Single cell sequencing data is available via the Gene Expression Omnibus database (GEO) under accession code GSE240064 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE240064].Data used for graphs are available in the Source Data file.All other data is available from authors upon request.
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